These mice are hypomorphic compound heterozygotes; the T1 HMB-synthase allele contains a neomycin gene with a bidirectional phosphoglycerate kinase promoter in exon Olaparib clinical trial 1 and the T2 allele has an alternative splice acceptor site in intron 1. Male T1/T2 mice (35�C45 days old) were intraperitoneally injected with 0.15 ml of saline solution, with or without rAAV2/8-HMBS. Urines (24 hour) were collected in metabolic cages. Phenobarbital induction was performed as previously described;10 however, the dose was increased to 110, 120, 125, 130 mg/kg/day for four consecutive days. For rotarod analysis, T1/T2 mice were trained for 3 days (two trials per day, 60 seconds maximum per trial) and tested on the forth day, at a rotation speed of 16 rpm (two trials per day, 180 seconds maximum) at 7 months of age.

Footprint analysis was performed at 9 months of age, as previously described.10 Analysis of variance was employed for statistical evaluation. Mice were killed at the indicated times by overdose injections of avertin and perfused with phosphate-buffered saline. Tissues from various organs were harvested and snap frozen in liquid nitrogen until use. HMB-synthase enzyme and porphyrin precursor assays. Tissues were weighed and three volumes/weight of chilled reporter lysis buffer (Promega, Valencia, WI) was added. Homogenization was performed on ice, using a glass homogenizer fitted with a pestle, at a speed of 145 rpm. The samples were centrifuged at 4 ��C at 15,000g until the supernatants were clear.

HMB-synthase enzyme assays were performed as previously described26 and protein concentrations were measured using the DC protein assay kit, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). One unit of enzymatic activity was defined as that amount of enzyme consuming 1 ��mol of PBG per hour. Urinary ALA and PBG levels were determined using the ALA/PBG column kit (Bio-Rad), whereas creatinine was measured using a colorimetric assay based on the picric acid method.27 DNA extraction and quantitation of AAV vector DNA. Total (genomic and plasmid) DNA was extracted from tissues of rAAV2/8-HMBS-treated AIP mice using the Puregene DNA kit (Qiagen). For each sample, 500 ng of total DNA was subjected to TaqMan real-time PCR using primers that specifically annealed to sequences of the murine HMB-synthase complementary DNA (forward primer: 5��-CGCCACCATGTCCGGTAA-3��, reverse primer: 5��-AGCATCGCCACCACAGTGT-3��) and a 6-FAM-TAMRA Anacetrapib labeled probe (5��-CGGCCACAACCGCGGAAGAA-3��). PCR conditions used were 50 ��C for 2 minutes, 95 ��C for 10 minutes, 40 cycles of 95 ��C for 15 seconds, 60 ��C for 1 minute, and vector copy numbers were quantitated with an ABI Prism 7900 sequence detection system.